Nontumorigenic mutants of Agrobacterium tumefaciens are being isolated and characterized with respect to the location of their genetic lesions and their ability to produce factors stimulatory to the growth of plant callus cultures. Mutations residing on the chromosome will be mapped by conjugation. In this regard derivatives of the R plasmid R68.45 which show enhanced sex factor activity are being developed by both in vivo and in vitro techniques. We are also isolating, by transposon insertion, auxotrophic mutants for mapping purposes. Mutations affecting Ti plasmid DNA will be mapped by complementation analysis. For this purpose we are developing pAgK84, the bacteriocinogenic plasmid from A. radiobacter strain K84, as a cloning vehicle for A. tumefaciens. Finally, we are analyzing our mutants for their ability to phenotypically complement one another in tumor induction. Using genetically marked complementing pairs, we will determine which member transfer its Ti plasmid DNA to the plant cell during tumor initiation.